Platelet membrane biomimetic nano-drug delivery system and preparation method and application thereof

Abstract

This invention provides a platelet membrane biomimetic nanomedicine delivery system, its preparation method, and its applications, relating to the field of pharmaceutical technology. This invention, through membrane engineering modification, utilizes a hybrid encapsulation material obtained from a platelet membrane and NK cell exosomes to load ginsenoside Rg3, resulting in a ginsenoside Rg3 delivery system with high encapsulation efficiency and high drug loading capacity. Furthermore, this system exhibits excellent stability and can be stored for extended periods at both room and high temperatures. The preparation method of this ginsenoside Rg3-loaded platelet membrane biomimetic nanomedicine delivery system is simple and suitable for widespread application. This ginsenoside Rg3-loaded platelet membrane biomimetic nanomedicine delivery system can effectively improve the therapeutic effect of ginsenoside Rg3 in lung cancer treatment, providing a novel drug for lung cancer treatment. Moreover, this drug has high safety and biocompatibility, exhibits no adverse reactions during treatment, and demonstrates significant anti-inflammatory effects.

Description

Technical Field

[0001] This application relates to the field of pharmaceutical technology, and in particular to a platelet membrane biomimetic nanodrug delivery system, its preparation method, and its application. Background Technology

[0002] Currently, the efficacy of many traditional drugs is severely limited due to poor pharmacokinetics, easy elimination from the body, and high cytotoxicity. In recent years, nanoparticle technology, especially nanoparticle drug delivery systems, has received widespread attention and is increasingly being applied in the medical field. Thanks to their small size, high targeting specificity, and long circulation time, nanoparticles are ideal drug carriers, which can improve in vivo targeting efficiency and reduce non-specific adverse reactions to some extent. However, despite the significant progress and attention given to nanoparticle drug delivery systems in the medical field, several key issues remain to be addressed. The stability of nanoparticle drug delivery systems, drug resistance, and the low target organ enrichment leading to unsatisfactory efficacy are problems that still need to be solved. Furthermore, the biocompatibility of nanoparticle drug delivery systems in vivo still needs improvement to meet the requirements of precise disease treatment.

[0003] Platelets are small fragments of cytoplasmic material shed from the cytoplasm of mature megakaryocytes in the bone marrow of mammals. Although platelets account for only 0.5%-1% of all blood cells, they play a crucial role in the body. Recent studies have shown that platelet membranes can serve as biomimetic coatings, endowing nanoparticles with the ability to avoid macrophage recognition, capture circulating tumor cells, and locate sites of inflammation, making them an ideal drug carrier. However, current research on platelet-based drug delivery is limited. Summary of the Invention

[0004] To address the above problems, this invention provides a platelet membrane biomimetic nanodrug delivery system, its preparation method, and its application.

[0005] On the one hand, this application provides a method for preparing a platelet membrane biomimetic nanodrug delivery system, the method comprising the following steps:

[0006] Step 1: Mix platelet membranes with cell exosomes and extrude to obtain hybrid material;

[0007] Step 2: Mix ginsenosides with hybrid materials, stimulate the fusion of ginsenosides and hybrid materials using electroporation, and incubate.

[0008] Furthermore, the exosomes are selected from one or more of MSC cell exosomes, NK cell exosomes, and 293T cell exosomes.

[0009] Preferably, the exosomes are NK cell exosomes.

[0010] Furthermore, the volume ratio of the platelet membrane to the exosomes is 1:(0.5-2).

[0011] The volume ratio of platelet membrane to exosomes can be any value among 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, and 1:2.

[0012] Preferably, the volume ratio of the platelet membrane to the exosomes is 1:1.

[0013] Preferably, the extrusion conditions are 10-30 extrusions of a 100-200 nm filter membrane.

[0014] More preferably, the extrusion conditions are 20 extrusions of a 100 nm filter membrane.

[0015] Furthermore, the ginsenosides are selected from one or more of ginsenoside Rg3, ginsenoside Rb1, ginsenoside Rb2, and ginsenoside Rb3.

[0016] Preferably, the ginsenoside is ginsenoside Rg3.

[0017] Furthermore, the concentration of the ginsenoside is 0.1 mg / mL to 0.3 mg / mL.

[0018] The concentration of the ginsenosides can be any value among 0.1 mg / mL, 0.2 mg / mL, and 0.3 mg / mL.

[0019] Preferably, the concentration of the ginsenoside is 0.1 mg / mL.

[0020] In a preferred embodiment, the solvent for the ginsenosides is water.

[0021] Furthermore, the volume ratio of the ginsenosides to the hybrid material is 1:(1-3).

[0022] The volume ratio of ginsenosides to hybrid materials can be any value among 1:1, 1:2, and 1:3.

[0023] Preferably, the volume ratio of ginsenoside to hybrid material is 1:2.

[0024] Furthermore, the conditions for stimulating the fusion of ginsenosides with hybrid materials by electroporation include: voltage 300-400V / cm, and number of electric shocks 2-4 times.

[0025] The voltage can be any value among 300 V / cm, 310 V / cm, 320 V / cm, 330 V / cm, 340 V / cm, 350 V / cm, 360 V / cm, 370 V / cm, 380 V / cm, 390 V / cm, and 400 V / cm.

[0026] The number of electric shocks can be any value of 2, 3, or 4.

[0027] Preferably, the voltage is 350 V / cm, and the electric shock is performed 3 times.

[0028] Preferably, each electric shock lasts 10-30 ms, with an interval of 1-2 s.

[0029] More preferably, each electric shock lasts for 10 ms, with an interval of 1 s.

[0030] Preferably, the incubation conditions include shaking incubation at 36°C-38°C for 1-3 hours.

[0031] More preferably, incubate at 37°C with shaking for 1 h.

[0032] In a preferred embodiment, the method includes the following steps:

[0033] Step 1: Mix platelet membranes and NK cell exosomes at a volume ratio of 1:(0.5-2), and extrude the mixture 10-30 times through a 100-200 nm filter membrane using a micro extruder to obtain the hybrid material;

[0034] Step 2: Dissolve ginsenoside Rg3 in water. Mix 0.1 mg / mL to 0.3 mg / mL of ginsenoside Rg3 with the hybrid material at a volume ratio of 1:(1-3). After mixing, apply an electric shock at 300-400 V / cm for 2-4 times, each time for 10-30 ms, with an interval of 1-2 s. Stimulate the fusion using electroporation. Incubate at 36°C-38°C with shaking for 1-3 h to obtain a biomimetic nanomedicine delivery system for platelet membrane-encapsulated ginsenoside Rg3.

[0035] Preferably, the method further includes a purification step.

[0036] Those skilled in the art can use common methods to purify it.

[0037] In a preferred embodiment, the purification includes: after incubation, adding an anticoagulant buffer solution, centrifuging at room temperature for 10-30 min at 0.5-1 g, discarding the supernatant, and repeating 1-3 times to remove free ginsenoside Rg3 and solvent.

[0038] On the other hand, this application also provides a platelet membrane biomimetic nanodrug delivery system prepared by the method described herein.

[0039] Preferably, the platelet membrane biomimetic nanomedicine delivery system can achieve an encapsulation rate of ginsenosides of over 80%; more preferably, it can achieve 82.04%.

[0040] Furthermore, the ginsenosides are selected from one or more of ginsenoside Rg3, ginsenoside Rb1, ginsenoside Rb2, and ginsenoside Rb3.

[0041] Preferably, the ginsenoside is ginsenoside Rg3.

[0042] Preferably, the platelet membrane biomimetic nanodrug delivery system has good stability and can be stored at 60°C for more than 30 days.

[0043] On the other hand, this application also provides a composition comprising the aforementioned platelet membrane biomimetic nanodrug delivery system.

[0044] The compositions described in this application may also contain excipients, which may be suitable solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration promoters, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, release inhibitors, etc.

[0045] Preferably, the composition can be prepared as an injection solution.

[0046] The compositions described in this application can be prepared by common methods, wherein one or more diluents or carriers may be added.

[0047] On the other hand, this application also provides the application of the platelet membrane biomimetic nanodrug delivery system or the composition thereof in the preparation of lung cancer drugs.

[0048] Preferred results show that the tumor inhibition rate for lung cancer can reach over 68%.

[0049] Preferably, the platelet membrane biomimetic nanodrug delivery system has high safety, high biocompatibility, and a low adverse reaction rate during treatment.

[0050] Preferably, the defect rate is less than 10%, and more preferably, 6.89%.

[0051] Preferably, the platelet membrane biomimetic nanodrug delivery system or the composition also has an anti-inflammatory effect.

[0052] The present invention has the following beneficial effects:

[0053] 1. In this application, ginsenoside Rg3 was loaded onto a coating material obtained by membrane engineering by hybridizing platelet membrane and NK cell exosomes for the first time, resulting in a ginsenoside Rg3 delivery system with high encapsulation efficiency and high drug loading capacity. Furthermore, the system has excellent stability and can be stored for a long time in ambient and high temperature environments.

[0054] 2. The preparation method of this platelet membrane biomimetic nanomedicine delivery system loaded with ginsenoside Rg3 is simple and suitable for widespread application;

[0055] 3. This platelet membrane biomimetic nanomedicine delivery system loaded with ginsenoside Rg3 can effectively improve the therapeutic effect of ginsenoside Rg3 in lung cancer treatment, providing a new type of drug for lung cancer treatment. Moreover, this drug has high safety and biocompatibility, no adverse reactions during treatment, and significant anti-inflammatory effects. Detailed Implementation

[0056] To more clearly illustrate the overall concept of this application, a detailed description is provided below by way of embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described to avoid confusion with the invention.

[0057] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the respective manufacturers.

[0058] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0059] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0060] Unless otherwise specified, in the following embodiments, reagents or instruments whose manufacturers are not indicated are all conventional products that can be purchased commercially.

[0061] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques from the fields of microbiology, biochemistry, analytical chemistry, pharmacy, zoology, medicine, cell culture, and related fields.

[0062] In addition, the "water" mentioned in this invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion-exchanged water, double-distilled water, high-purity water, and purified water.

[0063] In the following embodiments, unless otherwise specified, % means wt%, i.e., weight percentage.

[0064] Those skilled in the art can obtain platelet membranes using common methods. In a preferred embodiment, the platelet membrane preparation method includes: collecting blood from the abdominal aorta of adult SD rats; centrifuging 200 g of the collected whole blood for 20 min, collecting the supernatant, centrifuging at 1800 g for 20 min at room temperature, washing the precipitate repeatedly 3 times, and then centrifuging at 1800 g for 5 min to obtain platelets. The platelets are then incubated in a constant temperature water bath at 43℃ for 30 min, followed by repeated freeze-thaw cycles 3 times, centrifuged at 8000 g for 15 min at room temperature, the supernatant is removed, washed, resuspended, and sonicated at 20-25 kHz for 3 min using a water bath sonicator to obtain the platelet membrane.

[0065] MSC Cell Exosomes (Human) (Catalog No. 41220ES60), NK Cell Exosomes (Human) (Catalog No. 41221ES60), and 293T Cell Exosomes (Catalog No. 41219ES60) were purchased from Yisheng Biotechnology.

[0066] Ginsenoside Rg3 (CAS No.: 14197-60-5), ginsenoside Rb1 (CAS No.: 41753-43-9), ginsenoside Rb2 (CAS No.: 11021-13-9), and ginsenoside Rb3 (CAS No.: 68406-26-8) were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0067] Example 1: Preparation of a biomimetic nanomedicine delivery system for platelet membrane-encapsulated ginsenosides

[0068] Platelet membranes can serve as biomimetic coatings, enabling nanoparticles to evade macrophage recognition, capture circulating tumor cells, and locate sites of inflammation. However, due to the large molecular weight of ginsenosides, the encapsulation efficiency of platelet membranes alone is limited. In this embodiment, the preparation method of platelet membrane-encapsulated ginsenosides was optimized and screened through membrane engineering to improve its drug loading capacity and efficiency, as follows.

[0069] Step 1: Mix platelet membranes and cell exosomes (MSC cell exosomes, NK cell exosomes, 293T cell exosomes) at a 1:1 volume ratio, and extrude the mixture 20 times using a micro-extruder with a 100 nm filter membrane to obtain hybrid material;

[0070] Step 2: Ginsenosides (ginsenoside Rg3, ginsenoside Rb1, ginsenoside Rb2, and ginsenoside Rb3) were dissolved in water. 0.1 mg / mL of ginsenosides was mixed with the hybrid material at a 1:1 volume ratio. After mixing, electroporation was performed twice at 300 V / cm, each time for 10 ms with a 1 s interval, to stimulate fusion. The mixture was then incubated at 37°C with shaking for 1 h. After incubation, an anticoagulant buffer was added, and the mixture was centrifuged at room temperature (0.8 g) for 20 min. The supernatant was discarded, and this process was repeated three times to remove free ginsenosides and solvent, thus obtaining a biomimetic nanomedicine delivery system for platelet membrane-encapsulated ginsenosides.

[0071] The encapsulation efficiency (EE) of a biomimetic nanomedicine delivery system containing platelet membrane-encapsulated ginsenosides was determined using an enzyme-linked immunosorbent assay (ELISA). The method involved measuring absorbance at the maximum absorption wavelength of 540 nm, constructing a standard curve by performing a linear regression with absorbance as the ordinate and concentration as the abscissa, and measuring the concentration of unbound drug in the supernatant. The encapsulation efficiency was calculated based on the initial concentration of the drug solution and the concentration of unbound drug in the supernatant, using the formula below. The results are shown in Table 1.

[0072] EE (%) = (initial concentration of drug solution - concentration of supernatant) / initial concentration of drug solution × 100%.

[0073] Table 1

[0074]

[0075] As shown in Table 1, the encapsulation effect of ginsenoside Rg3 was optimal when using a hybrid material of NK cell exosomes and platelet membranes, achieving an encapsulation rate of over 70%. In this embodiment, parameters such as the volume ratio of platelet membrane to exosomes, the volume ratio of ginsenosides to the hybrid material, voltage, and number of electrotherapy shocks were further screened and adjusted. The results are shown in Table 2.

[0076] Table 2

[0077]

[0078] As shown in Table 2, the optimized platelet membrane-encapsulated ginsenoside biomimetic nanomedicine delivery system achieves an encapsulation efficiency of over 80%. This method also effectively improves the drug loading capacity of ginsenoside Rg3 and its solubility in the product.

[0079] In summary, the preparation method of the optimized platelet membrane-encapsulated ginsenoside biomimetic nanomedicine delivery system includes the following steps:

[0080] Step 1: Mix platelet membranes and NK cell exosomes at a 1:1 volume ratio, and extrude the mixture 20 times using a micro-extruder through a 100 nm filter membrane to obtain the hybrid material.

[0081] Step 2: Dissolve ginsenoside Rg3 in water. Mix 0.1 mg / mL ginsenoside Rg3 with the hybrid material at a volume ratio of 1:2. After mixing, electroporate three times at 350 V / cm for 10 ms each time, with a 1 s interval, to stimulate fusion. Incubate at 37°C with shaking for 1 h. After incubation, add anticoagulant buffer solution, centrifuge at room temperature (0.8 g) for 20 min, discard the supernatant, and repeat three times to remove free ginsenoside Rg3 and solvent, thus obtaining a biomimetic nanomedicine delivery system for platelet membrane-encapsulated ginsenoside Rg3.

[0082] The following experiments were conducted using an optimized biomimetic nanomedicine delivery system that encapsulates ginsenoside Rg3 on platelet membranes as a sample.

[0083] Example 2 Stability Evaluation

[0084] In this embodiment, stability testing is performed on the sample optimized in Example 1.

[0085] 1. Encapsulation efficiency stability test:

[0086] In this embodiment, the biomimetic nanomedicine delivery system sample of platelet membrane-encapsulated ginsenoside Rg3 obtained in Example 1 was placed in physiological saline, and the dissolution of ginsenoside Rg3 at different time points (0 h, 12 h, 24 h) was observed. The concentration of ginsenoside Rg3 in the supernatant was detected by an enzyme-linked immunosorbent assay (ELISA) reader, and the concentration of ginsenoside Rg3 stably encapsulated in the platelet delivery system was calculated. The stability of in vitro encapsulation was then observed, and the rate of change of encapsulation efficiency was calculated. The method for calculating the encapsulation efficiency was the same as in Example 1, and the formula for calculating the rate of change is as follows. The results are shown in Table 3.

[0087] Rate of change (%) = (Initial encapsulation ratio - Final encapsulation ratio) / Initial encapsulation ratio * 100%

[0088] Table 3 Stability Results

[0089]

[0090] As shown in Table 3, the encapsulation efficiency of the samples did not change significantly, proving that the sample system was stable.

[0091] 2. Storage stability test:

[0092] In this embodiment, the biomimetic nanomedicine delivery system sample of platelet membrane-encapsulated ginsenoside Rg3 obtained in Example 1 was prepared into a lyophilized powder, which was then dispensed into sealed bottles (0.5 g / bottle). The sample was stored at 60°C for 30 days. After 30 days, its morphology was observed, and a comparison of the morphology before and after the experiment was made. The results showed that its morphology did not change before and after storage, proving that the sample has good stability.

[0093] Example 3: Experiment on the therapeutic effect of lung cancer

[0094] In this embodiment, the biomimetic nanomedicine delivery system for platelet membrane-encapsulated ginsenoside Rg3, optimized in Example 1, was used as a sample for lung cancer treatment testing. The method includes the following steps:

[0095] 5×10 6 LLC (mouse lung cancer cell) cells were subcutaneously inoculated into the right inner thigh of BALB / c mice. The presence of a tumor indicated successful modeling. Tumor size was assessed daily using calipers, and tumor volume was calculated. Tumors were considered successfully modeled when they reached 90-100 mm². 2Treatment began at a specific time. Mice were randomly assigned to groups, and the dose of ginsenoside Rg3 in the sample was 25 mg / kg, administered once every two days for a total of four doses. An equal volume of commercially available ginsenoside Rg3 injection was used as a positive control, and an equal volume of physiological saline was used as a negative control. After the experiment, the mice were euthanized by cervical dislocation. The mice were then quickly dissected, and tumor tissue was collected to calculate tumor volume and adverse reactions, including but not limited to weight loss, death, and severe diarrhea. The formulas for calculating tumor inhibition rate and adverse reaction rate are as follows, and the results are shown in Table 4.

[0096] Tumor inhibition rate (TGI)% = (1-T / C)×100, where T represents the tumor volume of the experimental group and C represents the tumor volume of the negative control group;

[0097] Adverse reaction rate % = (Number of mice with adverse reactions ÷ Total number of mice treated) × 100%.

[0098] Table 4

[0099]

[0100] As shown in Table 4, the biomimetic nanomedicine delivery system for platelet membrane-encapsulated ginsenoside Rg3, optimized in Example 1, exhibits significant tumor-suppressive effects, providing a novel drug for lung cancer treatment. Furthermore, it demonstrates high safety with a significantly reduced adverse reaction rate.

[0101] Example 4: Anti-inflammatory effect experiment

[0102] In this embodiment, IL-6 levels were detected in each group of mice in Example 3 using an ELISA kit, and the results are shown in Table 5.

[0103] Table 5

[0104]

[0105] As shown in Table 5, the biomimetic nanomedicine delivery system for platelet membrane-encapsulated ginsenoside Rg3 optimized in Example 1 has anti-inflammatory effects.

[0106] Example 5: In vivo biocompatibility evaluation

[0107] In this embodiment, in order to investigate whether the biomimetic nanomedicine delivery system of platelet membrane-encapsulated ginsenoside Rg3 optimized in Example 1 would damage organs, the heart, liver, spleen, lung, and kidney of the experimental mice in Example 3 were collected and H&E sections were observed. The results of H&E sections showed that the physiological structure of the above organs was normal, indicating that the delivery system has good safety and biocompatibility.

[0108] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. A method for preparing a platelet membrane biomimetic nanomedicine delivery system, characterized in that, The method includes the following steps: Step 1: Mix platelet membranes and NK cell exosomes at a volume ratio of 1:(0.5-2), and extrude the mixture 10-30 times through a 100-200 nm filter membrane using a micro extruder to obtain the hybrid material; Step 2: Dissolve ginsenoside Rg3 in water. Mix 0.1 mg / mL to 0.3 mg / mL of ginsenoside Rg3 with the hybrid material at a volume ratio of 1:(1-3). After mixing, apply an electric shock at 300-400 V / cm for 2-4 times, each time for 10-30 ms, with an interval of 1-2 s. Stimulate the fusion using electroporation. Incubate at 36°C-38°C with shaking for 1-3 h to obtain a biomimetic nanomedicine delivery system for platelet membrane-encapsulated ginsenoside Rg3.

2. The platelet membrane biomimetic nanodrug delivery system prepared by the method of claim 1.

3. A composition, characterized in that, The composition includes the platelet membrane biomimetic nanodrug delivery system as described in claim 2.

4. The use of the platelet membrane biomimetic nanomedicine delivery system as described in claim 2 or the composition as described in claim 3 in the preparation of lung cancer drugs.

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